ARTICLE
Gold Nanorods for Ovarian Cancer Detection with Photoacoustic Imaging and Resection Guidance via Raman Imaging in Living Mice Jesse V. Jokerst,† Adam J. Cole,† Dominique Van de Sompel,† and Sanjiv S. Gambhir*,†,‡ †
Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University, 318 Campus Drive, Stanford, California 94305-5427, United States, and ‡Bioengineering, Materials Science & Engineering, Bio-X, Stanford University, Stanford, California 94305, United States
ABSTRACT Improved imaging approaches are needed for ovar-
ian cancer screening, diagnosis, staging, and resection guidance. Here, we propose a combined photoacoustic (PA)/Raman approach using gold nanorods (GNRs) as a passively targeted molecular imaging agent. GNRs with three different aspect ratios were studied. Those with an aspect ratio of 3.5 were selected for their highest ex vivo and in vivo PA signal and used to image subcutaneous xenografts of the 2008, HEY, and SKOV3 ovarian cancer cell lines in living mice. Maximum PA signal was observed within 3 h for all three lines tested and increased signal persisted for at least two days postadministration. There was a linear relationship (R2 = 0.95) between the PA signal and the concentration of injected molecular imaging agent with a calculated limit of detection of 0.40 nM GNRs in the 2008 cell line. The same molecular imaging agent could be used for clear visualization of the margin between tumor and normal tissue and tumor debulking via surface-enhanced Raman spectroscopy (SERS) imaging. Finally, we validated the imaging findings with biodistribution data and elemental analysis. To the best of our knowledge, this is the first report of in vivo imaging of ovarian cancer tumors with a photoacoustic and Raman imaging agent. KEYWORDS: photoacoustic imaging . Raman imaging . SERS . SERRS . gold nanorod . ovarian cancer
E
arlier detection of ovarian cancer (OvCA) should eventually be possible and may significantly improve patient survival times.14 Patients diagnosed with early stages of OvCA when the disease is confined to the ovary have a five-year survival of up to 95% following conventional therapy. In late stages, however, the 5-year survival is a dismal 2530%.5 Thus, a convenient and accurate method to quickly and accurately identify affected individuals in the early stages of disease could be highly beneficial. There are two main approaches to the early detection of OvCA: in vitro blood-based biomarker testing including CA125 and HE4 and in vivo imaging.5 Realistic clinical solutions will likely require the strengths of both approaches, and work on serum assays continues elsewhere.69 Here, we describe improvements in imaging. Computed tomography (CT) may have utility for tumor staging, but offers poor JOKERST ET AL.
soft tissue contrast with resulting poor sensitivity and specificity in screening.5,10 Magnetic resonance imaging (MRI) offers excellent contrast without ionizing radiation, but has temporal and financial needs that are likely inconsistent with high throughput screening.5 Positron emission tomography (PET) with a very high sensitivity can interrogate various molecular/biochemical properties, but is more suitable for monitoring response to therapy than detecting early lesions due to limited spatial resolution.5,11 In contrast, ultrasound (US) imaging is real time, affordable, and offers clinical sensitivity near 90% in select patient cohorts;5 transvaginal US, transabdominal US, or both are currently considered the first-line imaging tool(s) whenever an ovarian lesion is suspected.5,12 Ultrasound can likely be well complemented by photoacoustic (PA) imaging.1318 In PA imaging, a short light pulse incident on the target tissue causes rapid VOL. 6
’
NO. 11
’
* Address correspondence to
[email protected]. Received for review September 19, 2012 and accepted October 27, 2012. Published online October 28, 2012 10.1021/nn304347g C 2012 American Chemical Society
10366–10377
’
2012
10366 www.acsnano.org
JOKERST ET AL.
with inductively coupled plasma spectrometry. To the best of our knowledge, this is the first report of in vivo imaging of OvCA tumors with a PA imaging agent and the first report of a combined PA/SERS imaging probe. RESULTS GNR Physical Characteristics and Signaling Capabilities. GNRs with different resonances were physically characterized and analyzed for their capacity to produce both SERS and PA signal.34,44 The size of the GNRs was tuned by changing the concentration of silver nitrate.44 Three batches were selected for further study;GNRs with peak absorbance at 661, 698, and 756 nm. TEM imaging confirmed the expected morphology (Figure 1A and Supporting Information Figure S.1) and indicated that 15% of each batch was not composed of GNRs, but rather gold particles with assorted morphology including gold nanosquares, nanospheres, and nanopolyhedrons. The width and length (and thus aspect ratio) of at least 30 different GNRs from multiple TEM fields-of-view were measured using ImageJ software. Absorbance spectroscopy (Supporting Information Figure S.2A) indicated that higher aspect rods had more red-shifted absorbance peaks,45,46 and PA analysis confirmed that the wavelengths of highest absorbance correlated to the highest PA signal (Supporting Information Figure S.2B). A blue shift of approximately 510 nm from peak resonance was sometimes observed in the first week after synthesis, similar to other reports.47 GNRs were dialyzed with an IR laser dye and thiol-PEG versus distilled water to remove cetyltrimethylammonium (CTAB) and increase SERS signal. Zeta potential in 1:1 water/PBS before and after functionalizing with IR792 and thiol-PEG showed a consistent shift away from a positive zeta potential (due to quarternary ammonium on CTAB) to a more neutral value when PEG-coated (Table 1). Different dyes produce different SERS spectra as shown in Figure 1B. Each GNR type in Figure 1B has unique peaks that could be easily spectrally unmixed from a combination of the three for multiplexed analysis. These peaks include 934 and 1206 cm1 for IR792, 789, 852, and 1080 cm1 for DTTC, and 1310 and 1433 cm1 for IR140. The data in Figure 1B are normalized relative to each species' maximum absorbance; however, the three dyes produced very different data on an absolute scale. The IR792 dye produced the highest signal. Other molecules were at lower intensity: DTTC (16.2%; 849 cm1), IR140 (9.9%; 1312 cm1), IR1061 (0.48%; 1558 cm1), and Coumarin (0.27%; 1038 cm1). The number in parentheses indicates the signal intensity at the maximum peak relative to IR792 and the peak of dye maximum signal. IR792 functionalized GNRs were used for the remainder of the experiments. The 661, 698, and 756 nm GNRs (100 μL at 5.4 nM) were studied using PA imaging with laser excitation at 680, 698, and 756 nm, respectively (Table 2). The VOL. 6
’
NO. 11
’
10366–10377
’
ARTICLE
heating and thermal expansion that produces a pressure wave capable of acoustic detection. PA imaging produces a tomographic image in vivo with very high spatial resolution (up to 50500 μm) and depth of penetration up to 5 cm. PA imaging can use endogenous contrast like hemoglobin and melanin or a variety of exogenous imaging agents including small molecules and nanoparticles. Here, the former offers better tissue access and the latter produces significantly more signal on a mole-to-mole basis.19,20 Common nanoparticle PA imaging agents include carbon nanotubes, copper sulfide, and iron oxide, with detection limits in the nM range depending on instrumentation.18,21,22 Finally, Raman imaging is a next generation optical technique that uses noble metal nanoparticles for surface-enhanced Raman spectroscopy (SERS) or surface enhanced resonance Raman spectroscopy (SERRS), resulting in highly sensitive (fM) and multiplexed (n > 5) imaging.2329 The current work combines the attributes of PA and SERS imaging into a single multimodal imaging agent via gold nanorods (GNRs). While we have previously used gold spheres for both PA and SERS,30 GNRs have several advantages that motivate this work.3133 They have a sufficiently large optical absorption cross section to maximize the agent's PA signal, yet are small enough to delay/minimize uptake by the reticuloendothelial system. GNRs are easily functionalized with a variety of SERS reporters for multiplexing,34 and their tips offer higher SERS effects relative to spheres.35 GNRs may also better extravasate via the enhanced permeability and retention (EPR) effect into the tumor space versus spherical nanoparticles due to their higher aspect ratios.36,37 Furthermore, gold has been used clinically for decades,38 and GNRs likely lack much of the toxicity concerns that have hampered other PA imaging agents such as carbon nanotubes.39,40 Finally, GNRs have great potential as photothermal ablation tools that could be incorporated into a combined therapeutic/diagnostic or “theranostic” approach.31,34,4143 In this report, we prepare and characterize dual modality SERS and PA GNRs and use them to image ovarian tumor subcuatneous xenograft models in vivo. We envision using this combined imaging agent to evaluate ovarian lesions in at-risk patients first identified by blood-based screens. This imaging agent offers both PA signal for diagnostic or prognostic imaging studies and optical SERS signal for image-guided resection. The PA modality overcomes the SERS challenge of poor depth of penetration and the SERS modality compensates for PA limitations with sensitivity. Three different sizes of GNRs were studied with low in vitro toxicity and stable signaling capacity. The imaging agent easily imaged the xenograft tumors of three common OvCA cell lines: 2008, HEY, and SKOV3. Tumor margins were clearly visualized optically with the SERS modality, and the imaging data was validated
10367
2012 www.acsnano.org
ARTICLE Figure 1. Gold nanorods for PA and SERS Imaging. (A) TEM image of GNRs shows typical morphology with an aspect ratio of 3.5. (B) After functionalization with different Raman reporter molecules, a unique spectrum is recorded for each type of GNR. Each spectrum is normalized to its own maximum value. (C) The GNRs have both a longitudinal (∼760 nm) and an axial (∼530 nm) absorbance peak. (D) A comparison of the SERS signal of GNRs (left ordinate) to a similar silica-coated spherical nanoparticle (right ordinate) shows enhanced signaling capacity, but with higher baseline for the GNRs. In panel D, both spectra are collected from a 17 pM sample, 1 s acquisition time, and a 12 objective. a
TABLE 1. Physical Characterization of Test GNR Batches GNR absorbance max
length
width
aspect
(nm)
(nm)
(nm)
ratio
661 698 756
43.6 ( 6.0 18.0 ( 2.1 44.5 ( 3.6 15.4 ( 1.7 41.4 ( 3.7 12.0 ( 1.7
2.4 2.9 3.5
zeta (mV)
zeta (mV)
(before PEG) (after PEG)
þ14.6 þ26.4 þ29.3
2.5 0.1 8.9
a
The optical resonance of the GNRs becomes increasingly red-shifted as the aspect ratio increases. Exchanging the CTAB surfactant for PEG neutralizes the zeta potential for all three batches.
756 nm GNRs produced the most intense signal; 698 GNRs were most intense when normalized to laser intensity. Unfortunately, the highest absorbance peak of the 661 nm GNRs could not be used because the lower range of the laser is 680 nm. All batches had similar SERS signal using the IR792 dye (Table 2) and 785 nm excitation. If two more rounds of centrifugation and washing were done during the postdialysis purification, we noticed visible aggregates that could be only be resuspended with ultrasonication. Although visibly resuspended, TEM imaging, absorbance spectroscopy, and SERS analysis indicated that some residual aggregation remained (Supporting Information Figure S.3). The formation of “hotspots” between the nanoparticles increased the SERS effect 2-fold versus monodisperse GNRs.48,49 The ex vivo LOD of the GNRs was determined in both the SERS (Supporting Information Figure S.4) and JOKERST ET AL.
PA (Supporting Information Figure S.5C) modalities. The in vitro SERS detection limit was 17 fM; the in vivo SERS detection limit was 8.5 pM (Supporting Information Figure S.4). The SERS intensity of the GNRs was compared to silica-coated gold nanospheres at 17 pM (Figure 1D) described previously.23,25,50,51 The results indicate that the GNRs produce nearly a log order more signal on a per particle basis. In the PA modality, the ex vivo LOD of the 756 nm GNRs is 24 pM (Supporting Information Figure S.5B). The batch-to-batch signal reproducibility of four different batches of GNRs was studied with a relative standard deviation of 15.5% and 3.6% for the SERS and PA modalities, respectively. Probe Stability and Toxicity. GNR stability was tested in water and murine serum. GNRs were brought to 0.13 nM and analyzed longitudinally over 30 h. There was no decrease in the probe SERS intensity in water, but nearly a doubling of SERS signal in serum. This observation was present in repeated experiments and attributed to aggregation of the GNRs causing SERS “hot spots” that further enhance the SERS signal.48 Toxicity was studied in a pilot fashion with a modified version of the Alamar Blue assay (Presto Blue).52 We first confirmed the ability of the reagent to measure metabolic activity of the OvCA cells lines 2008, HEY, and SKOV3 using increasing concentrations of cells (Supporting Information Figure S.6). The correlation coefficient (R2) was above 0.97 for all three OvCA cell VOL. 6
’
NO. 11
’
10366–10377
’
10368
2012 www.acsnano.org
GNR max (nm)
PA Ex. λ (nm)
PA Ex. intensity (mJ)
PA signal normalized
Raman λ Ex. (nm)
Raman signal (arb unit)
661 698 756
680 698 756
3.2 4.0 6.3
56.6 99.2 115.9
785 785 785
3920 ( 260 1770 ( 330 2430 ( 390
a
The ex vivo PA signal of the three different classes of GNRs increases with resonance. PA samples analyzed at 2.2 nM and Raman analysis at 17 pM. When adjusted for the laser power, the 698 nm resonant GNRs offer the highest signal. The SERS signal for the three classes of GNRs is within the same order of magnitude. These data and the data in Supporting Information Figure S.8, suggested that 756 nm GNRs were most suitable for use in the remainder of the experiments.
types. In a separate plate, increasing concentrations of the 756 nm GNRs were added to 10 000 OvCA cells in replicate (n = 6) wells of a 96 well plate and allowed to incubate overnight. A decrease in metabolic activity was observed at 0.5 nM for 2008 cells; no toxicity up to 1 nM GNRs was observed for HEY and SKOV3 cells (Figure 2.). However, 1 nM of GNRs prior to exchange of PEG for CTAB had significantly (p < 0.05) higher toxicity for all three cell lines (Figure 2). PA Imaging. We envision using PA imaging as an accessory to B-mode ultrasound, and it would be used to identify and characterize OvCA tumors. To determine stability of PA signal we repeatedly imaged one tumor bearing mouse without any molecular imaging agents through three different sets of challenges: (1) no movement of the animal between scans, (2) removing and repositioning the mouse in between scans by the same operator, and (3) removing and repositioning the mouse in between scans by different operators. The relative standard deviation (RSD) for these three challenges is 9.6, 13.5, and 6.5%, respectively (Supporting Information Figure S.7A). Differences in absolute intensity are expected because not all experiments were done on the same day. We also studied whether any photothermal effects are observed during PA imaging and saw no temperature increase due to the relatively low laser power ( 0.999 (Supporting Information Figure S.12A). When decreasing volumes of 756 nm resonance GNRs were dissolved in aqua regia, the gold signal was also linear at R2 > 0.99 (Supporting Information Figure S.12B). We also spiked digested tissues with gold standard to 10 ppm and compared that signal to 10 ppm in aqua regia only. The signal in blood, tumor, spleen, liver, lung, and kidney was 104%, 103%, 106%, 109%, 106%, and 107% of the signal without digested tissues (Supporting Information Figure S.13A). Finally, to determine if analyte was lost in the sample preparation process, 50 μL of GNRs was added to mouse tissues, digested, analyzed, and the signal compared to the signal from 50 μL of GNRs digested with aqua regia only and no processing (Supporting Information Figure S.13B). Percent recovery values ranged from 96.7% for spleen to 100.9% in lung, which indicated nearly complete recovery of analyte and sample. For these reasons, we used the raw data collected from experimental samples (Figure 5) and did not apply any correction factors. Not surprisingly, there is longitudinal accumulation of the GNRs in organs of the reticuloendothelial system (RES) like the spleen and liver and a corresponding decrease in GNRs available in the blood pool (Figure 5A)56,57 (see also Supporting Information Table 1 for the raw gold content). Thirty minutes postinjection the blood pool contains 47.3 ( 5.0%ID/g of gold; this value decreases to 16.1 ( 2.1%ID/g 6 h postinjection. However, one key finding is that the tumor signal remains elevated (3 μg Au/g) at the 24 h time period, after the blood pool level has returned to baseline (0.2 μg Au/g) indicating retention of GNRs within the tumor bed. Importantly, there was a linear (R2 > 0.91) relationship between the PA signal measured in the tumor and the gold content in the tumor. Both values peaked at 3 h postinjection. Tumors were shown to have maximum gold content of 3 h with 3.80 μg of gold per gram of tissue (Supporting Information Table S.1). When converted to molar JOKERST ET AL.
concentrations of GNRs using a density of 1 g/mL tumor, this value is 29 pM, well above the in vivo and in vitro detection limits presented in Supporting Information Figure S.4. SERS Imaging. The GNRs have both PA and SERS signaling capacity, and the SERS imaging modality was used to discriminate tumor margins for imageguided resection 24 h after injection (Figure 6). A SERS signal was clearly present in all three OvCA tumor types. SERS maps were constructed by comparing the SERS spectrum at each point to a reference spectrum constructed from GNRs before injection through dynamic least-squares analysis. The intensity of the pixel is a measure of how well the collected spectrum matches the reference spectrum. Brighter pixels are more closely matched. This mapping shows that the brightest pixels correlate to the tumor (Figure 6A,B). In addition to illustrating tumor boundaries, the Raman signal can also be used for image-guided resection (Figure 7). The number of positive pixels in the tumor area decreased from 65.7% before resection (Figure 7A) to 6.2% after resection (Figure 7C). DISCUSSION We report a GNR molecular imaging agent with PA and SERS imaging capabilities and used this to image OvCA tumor models. To the best of our knowledge, this is the first example of a dual PA/SERS imaging with OvCA tumors. The GNR was stable with minimal toxicity in cell culture (Figure 2) and had detection limits (17 fM SERS, 24 pM PA) better than other reported PA (50 nM carbon nanotubes;21 50 pM gold spheres30) and SERS (gold core/silica shell) imaging agents (610 fM).23,24 Higher signal intensity of GNRs would be useful to generate high contrast or to generate a moderate amount of contrast with a low injected dose. The GNRs had an order more signal per mole versus the core/shell agents. The 120 nm silica shell/gold core particle previously described in our work has a volume of 106 nm3,51,58 VOL. 6
’
NO. 11
’
10366–10377
’
10371
2012 www.acsnano.org
ARTICLE Figure 6. Tumor margin identification with SERS imaging. (A) Photograph of mouse bearing a 2008 tumor (M = muscle; T = tumor; L = liver) with epidermis removed. Green box indicates region subjected to SERS imaging 24 h postinjection. (B) SERS imaging of area indicated in panel A shows increased SERS in the tumor (red). This SERS map was created by comparing the SERS spectra at different spatial locations (red curve in panels C and D) to a reference spectra (blue curve in panels C and D) collected ex vivo. A close match of sample and reference spectra (C) yields an intense pixel in the SERS map (tumor; white arrow, B). A poor match of the sample and reference spectra (D) yields a dim pixel (adjacent muscle; red arrow in B). Intensity bar in B represents the least-squares coefficient comparing reference and sample spectra (see Methods). Panels C and D present spectra normalized to maximum signal for each spectrum.
while the GNR has a volume of 104 nm3. Thus, this approach offers a 10-fold improvement in SERS signal versus alternative approaches, but using only 1% of the volume. (When comparing to the gold core only (1.1 105 nm3), this factor is 8.8% of the volume.) Surprisingly, the Raman reporters BPE and S403 that had previously shown excellent performance when used with silicacoated gold nanospheres had no utility with the GNRs.23,51 Other reports suggested 1,5-dimercaptonapthalene may be a suitable Raman dye for GNRs,59 but it produced no signal above the background of nonfunctionalized GNRs. This low signal is likely caused by the different surface chemistry of CTAB-functionalized GNRs versus citratestabilized spherical nanoparticles. GNRs with three different aspect ratios were studied, and the most red-shifted batch (aspect ratio = 3.5) was selected for in vivo imaging of OvCA tumors because it offered the highest in vivo signal in subcutaneous implants. This is likely due to better passage of the PA excitation light through tissue at 756 nm versus the maximum absorbance wavelengths of more blueshifted GNRs.60,61 Stronger PA excitation energy at 756 nm, despite potential concerns with increased scatter relative to absorption,62 may also contribute to a better in vivo signal. Nevertheless, the PA signal is highly reproducible (
